Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP‐deficient lung cancer

Abstract The aggressive nature and poor prognosis of lung cancer led us to explore the mechanisms driving disease progression. Utilizing our invasive cell‐based model, we identified methylthioadenosine phosphorylase (MTAP) and confirmed its suppressive effects on tumorigenesis and metastasis. Patients with low MTAP expression display worse overall and progression‐free survival. Mechanistically, accumulation of methylthioadenosine substrate in MTAP‐deficient cells reduce the level of protein arginine methyltransferase 5 (PRMT5)‐mediated symmetric dimethylarginine (sDMA) modification on proteins. We identify vimentin as a dimethyl‐protein whose dimethylation levels drop in response to MTAP deficiency. The sDMA modification on vimentin reduces its protein abundance but trivially affects its filamentous structure. In MTAP‐deficient cells, lower sDMA modification prevents ubiquitination‐mediated vimentin degradation, thereby stabilizing vimentin and contributing to cell invasion. MTAP and PRMT5 negatively correlate with vimentin in lung cancer samples. Taken together, we propose a mechanism for metastasis involving vimentin post‐translational regulation.


9th Dec 2021 1st Editorial Decision
Dear Dr. Yu, Thank you for the submission of your research manuscript to our journal, which was now seen by three referees, whose reports are copied below.
We concur with the referees that the proposed role of PRMT5 mediated vimentin demethylation in MTAP deficient cancer cells is in principle very interesting. However, referees, especially referee #1, raise significant concerns that need to be addressed to consider publication here. I find the reports informed and constructive, and believe that addressing the concerns raised will significantly strengthen the manuscript. As the reports are below, and I think all points need to be addressed, I will not detail them here.
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I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if you have questions or comments regarding the revision. In this study, the group led by Sung-Liang Yu show that MTAP loss promotes invasion and metastasis in long cancer. Mass Spec. studies identified vimentin as a PRMT5 substrate that displays decreased SDMA in the absence of MTAP. MTAP-loss or PRMT5 knockdown results in vimentin stabilization, suggesting that the SDMA marking of vimentin promotes its destabilization. Indeed, the authors go on so show that vimentin becomes highly ubiquitinated in a SDMA-dependent manner. Finally, they show that MTAP/PRMT5 levels negatively correlated with vimentin levels in lung cancer samples.

Critique:
The key findings in this study are; First, is that MTAP loss promotes metastasis. Second, PRMT5 methylates vimentin. Third, vimentin is destabilized by PRMT5 methylation. Each of these findings are significant, but a number of points are still preliminary and addition experiments are needed to strengthen the overall hypothesis, before this study is suitable for publication in EMBO Reports.
The following needs to be addressed: 1. In Figure 2, the Western blot evidence showing vimentin is a PRMT5 substrate is very weak. Figure 2C needs to be repeated. This is a key figure, and all you see is a smudge using the SYM10 antibody. Also, the decrease in SDMA signal after PRMT5 siRNA (shown in Figure 2D) is not convincing. Ideally, the in vitro methylation experiment should be performed using radioactive AdoMet. Known substrates for PRMT5 (like SmB or histones) should be used as a positive control, which will allow us to determine how good a PRMT5 substrate vimentin is, compared to know PRMT5 targets.
2. According the "Material and Methods" section, the SDMA antibody used in the study was Sym10. This antibody was raised against a GAR motif (an RGG repeat). The 4 vimentin methylation sites identified in Figure 3 do not harbor GAR motifs. Thus, additional studies are needed to confirm that these four sites are indeed key PRMT5 targets. Two independent approaches should be taken here: 1) does the Sym10 antibody recognize peptides that harbor SDMA peptides for R196, R207, R345 and R364? The non-methylated peptide controls should not be recognized by this antibody. This experiment can be performed as a dot-blot. 2) Ideally, the authors will raise specific antibodies to the four PRMT5 sites.

In Figures 4 & 5, the authors introduce different mutations (K & F) into the vimentin methylation motifs.
They refer to the R-to-F mutation as a methyl-memetic. However, the R-to-F mutation is not a methyl-memetic, even though there are a few publications that refer to it as such. Here I will highlight the reasons why the R-to-F mutation is not a methyl-memetic: a. In the first place, methylated arginine residues retain a full positive charge on the side chain -there are no charges on the phenylalanine side chain. b. In the MMA side chain there are four hydrogen bond donors and in the ADMA/SDMA side chain there are three hydrogen bond donors -there are none in the phenylalanine side chain. c. Just in terms of carbon atoms, the phenylalanine side chain has seven; the MMA side chain has only five and the ADMA/SDMA side chain has only six. d. In terms of the overall number of non-hydrogen atoms, the phenylalanine side chain has seven, the MMA side chain has eight and the ADMA/SDMA has nine. e. There is no evidence that R-to-F mutation can be read by Tudor domains. So, the docking properties of the Rme reader domains are also not generated by this R-to-F mutation.
There is no need to remove the data that includes experiments with the R-to-F mutation as this mutant often behaves differently to the R-to-G and R-to-K mutants and does provide interesting data. However, all reference to the R-to-F mutation being a methyl-memetic should be removed from the text.
4. The emerging role of PRMT/proteosome crosstalk should be addressed in the discussion. There are a number of published studies related to this topic. Some works show that the SDMA PTM promotes protein stability, and others show that it stimulates ubiquitination.
5. The data in Figure 6 cannot be viewed. The authors should not use a black background for this figure.
6. Finally, is there a mechanistic explanation for the SDMA mark being a signal for protein degradation? Is there an E3 ligase that can read the SDMA marks on vimentin. This is a critical missing link in this study.
Referee #2: The authors identify the critical role MTAP/PRMT5 axis in post-transcriptional regulation of vimentin, which is a novel target for this post-translational modification/dimethylation. MTAP is responsible for catalyzing phosphorylation of MTA, so MTAP deficiency results in MTA accumulation, which reduces the level of PRMT5 (an arginine methyltransferase), which is required for demethylation of vimentin. This study uses methylproteomic screening to identify vimentin as novel dimethyl protein and cell and animal based assays with genetic knockout and post-transcriptional modification to test prove this mechanism of vimentin modification and its relevance to lung cancer progression. Overall, this study is very well written and the data very clearly illustrates the importance of their findings. In the abstract the authors say sDMA modification trivially affects the filamentous structure of vimentin and I would say this has not been fully proven, since only molecular methods were used to study vimentin structure. This is a small point that can be addressed through high resolution confocal microscopy experiments or other highresolution imaging or structural analysis.

Referee #3:
In this well-written and insightful manuscript by Chang et al., the authors investigated the repressive role MTAP plays in aggressive lung cancer and tumour models. It has been established that PRMT5 activity is negatively impacted by buildup of MTA when MTAP levels are low or absent. Through proteomics, they identified vimentin as a PRMT5 substrate; when vimentin is symmetrically methylated by PRMT5 on four unique residues, vimentin is degraded in the proteasome and is less likely to form filaments that promote cell invasion. The authors use several methods described in detail as to how MTAP/PRMT5 axis affects vimentin stability, and they highlight the importance of vimentin as a target and corroborate their findings by comparing overall survival in patients that are either MTAP high/ vimentin low or MTAP low/ vimentin high. This reviewer rates this manuscript as very high and impactful to PRMT and cancer fields as it represents a significant advance.
Very Minor: (1) Associated error for average fold change intensities for metabolites in Fig. S2a? (2) Bottom of page 11: A more accurate statement would be that the residue identity at these methylation sites governs the migration promotion of vimentin since sDMA is not being directly measured but correlated.
(3) "... nutrient-responsive PTMs on proteins or chromatins modulate..." chromatin without an "s" on page 18. (4) Top of page 19: "... the major type II PRMTs" PRMT without an "s" The following needs to be addressed: C1. In Figure 2, the Western blot evidence showing vimentin is a PRMT5 substrate is very weak. Figure 2C needs to be repeated. This is a key figure, and all you see is a smudge using the SYM10 antibody. Also, the decrease in SDMA signal after PRMT5 siRNA (shown in Figure  2D) is not convincing. Ideally, the in vitro methylation experiment should be performed using radioactive AdoMet. Known substrates for PRMT5 (like SmB or histones) should be used as a positive control, which will allow us to determine how good a PRMT5 substrate vimentin is, compared to know PRMT5 targets.

R1:
We would like to thank the reviewer for the constructive and helpful suggestions. In accordance with the suggestions, we have included histone H3 and H4 proteins as positive controls and utilized tritiated AdoMet as methyl donor in in vitro methylation assays. The fluorography data showed that vimentin can be methylated by PRMT5 even in the presence of dominant substrates, histone H3 and H4, albeit the signal is relatively weak due to the less amount of its input. These results suggest that vimentin serves as a favorable substrate of PRMT5. The corresponding Results (page 9) and Materials and Methods (page 27) were revised and the changes were indicated in blue.
Page 9, line 16 to line 22 To determine whether vimentin is a substrate of PRMT5, we performed an in vitro 20th Apr 2022 1st Authors' Response to Reviewers 2 methyltransferase assays in which the recombinant PRMT5 and vimentin were incubated with methyl donor S-adenosylmethionine (SAM). We found that vimentin was directly methylated by PRMT5 and the sDMA level of vimentin was mostly abolished by MTA, which is an analog of SAM and competitive inhibitor of methyltransferases (Kryukov et al., 2016) (Figs 2C and EV2E).
Revised Figure EV2E E. In vitro methylation of vimentin by PRMT5. Tritiated proteins were separated by SDS-PAGE, stained with Coomassie blue (left), dried and analyzed by fluorography (right). Histone H3 and H4 proteins were used as positive controls. *: tritiated vimentin.
Page 27, line 23 to Page 28, line 5 For fluorography, an in vitro methyltransferase assay using radioactive SAM (NET155V250UC, Perkin Elmer) was also carried out as follows. 1 μg PRMT5 and 1 μg substrate proteins (Histone H3, ab198757; Histone H4, ab198115; Abcam) were incubated in the presence of 3 µCi tritiated SAM (18.0 Ci/mmol from a 1.0 mCi/ml stock solution) for 1 hour at 30 o C in a final volume of 30 µl methylation reaction buffer. Subsequently, tritiated protein was separated by SDS-PAGE and then the gel was treated with EN 3 HANCE (6NE9701, Perkin Elmer) and exposed to a film at -80 o C for 7 days.
C2. According the "Material and Methods" section, the SDMA antibody used in the study was Sym10. This antibody was raised against a GAR motif (an RGG repeat). The 4 vimentin methylation sites identified in Figure 3

not be recognized by this antibody. This experiment can be performed as a dot-blot. 2) Ideally, the authors will raise specific antibodies to the four PRMT5 sites.
R2: We thank the reviewer for the insightful comments. To alleviate the concerns raised by the reviewer, we used synthetic di-methylated and non-methylated vimentin peptides to clarify the specificity of the SYM10 antibody by performing dot blot assays. We found that the dot intensity of sDMA from MTAP-knockout cell lysates (H1650 sgMTAP cell lysate) is weaker than that of the one from control cell lysates (H1650 sgCTRL cell lysate), consistent with the Western blot data ( Figure 2B). In addition, the full length vimentin recombinant protein used in in vitro methylation assays and non-methylated peptides (R196/R207 unmodified and R345/R364 unmodified) were not recognized by the SYM10 antibody, whereas synthetic di-methylated peptides (R196 me2s, R207 me2s, R196/R207 me2s, R345 me2s, R364 me2s, R345/R364 me2s) were reacted with the SYM10 antibody, supporting our notion that these four sites are key PRMT5 targets ( Figure 3). Indeed, we agree with the reviewer that generating specific methyl-vimentin antibodies would further confirm the four PRMT5 sites; however, it usually takes 4-6 months to produce a new antibody, and, in particular, we received synthetic peptides in the end of February in the wake of COVID-19; therefore, performing this additional study would have delayed this resubmission beyond the allotted time. The dot blot data were included in the Results section (page 11) and marked in blue.
Page 11, line 1 to line 2 The dimethylated forms of the four arginine residues can be recognized by an anti-sDMA antibody ( Fig EV3B).
Revised Figure EV3B

C3. In Figures 4 & 5, the authors introduce different mutations (K & F) into the vimentin methylation motifs. They refer to the R-to-F mutation as a methyl-memetic. However, the R-to-F mutation is not a methyl-memetic, even though there are a few publications that refer
to it as such. Here I will highlight the reasons why the R-to-F mutation is not a methyl-memetic: a. In the first place, methylated arginine residues retain a full positive charge on the side chain -there are no charges on the phenylalanine side chain.

There is no need to remove the data that includes experiments with the R-to-F mutation as this mutant often behaves differently to the R-to-G and R-to-K mutants and does provide interesting data. However, all reference to the R-to-F mutation being a methyl-memetic should be removed from the text.
R3: We would like to thank the reviewer for the comments. Yes, we agree that R-to-F mutant is not a perfect candidate to be a methyl-mimetic. The reason why researchers in previous studies used phenylalanine (F) is that phenylalanine (F) mutation introduced hydrophobicity, partially mimicking the bulky hydrophobic properties of methylated arginine residues. To better reflect the functionality of R-to-F mutant, we have removed the term "methylation-mimic" and revised the description for 4RF mutant in the Results section (pages 11 and 14), and have marked these changes in blue.
Page 11, line 3 to line 5 To verify whether these four arginines are the key dimethylation sites of vimentin, these arginines were individually or entirely mutated to lysines (non-arginine methylatable).

Page 11, line 15 to line 19
To investigate if the functionality of vimentin is mediated by its sDMA level, endogenous vimentin in H1650 and CL1-5 cells was knocked out by CRISPR/Cas9 approaches and the effect of wild-type, 4RK and 4RF (R196/207/345/364F, mimicking the bulky hydrophobic properties of methylated arginines) mutant vimentin on cell motility was evaluated.
Page 14, line 11 to line 13 As shown in Fig 5F,  In addition to the interplay with metabolites, the crosstalk between PTMs also influences the signal transduction and biological functions. Recently, there are a number of studies focusing on protein methylation-dependent ubiquitination and degradation. After being methylated by methyltransferase, the stability of substrate proteins would be either upregulated or downregulated, thereby strengthening or alleviating the involved pathways and functions such as alterations in lipogenesis, angiogenesis, metastasis, genome stability and carcinogenesis (Hu et al, 2015;Kim et al, 2016;Leng et al, 2018;Li et al, 2020;Liu et al, 2020;Liu et al, 2016). Therefore, protein methylation appears to be a new hallmark for protein stability.
C5. The data in Figure 6 cannot be viewed. The authors should not use a black background for this figure.
R5: We thank the reviewer for bringing this to our attention. We downloaded the files we previously submitted from the EMBO submission system and checked the content. The Figure 6 can be viewed as a TIF file. To address the concerns, we have re-exported the Figure 6 and pasted a copy below. D. An association between MTAP and vimentin levels in 124 lung cancer patients was examined by Spearman's rank correlation. E. Kaplan-Meier analyses of overall survival for lung cancer patients grouped by MTAP and vimentin levels from IHC staining. p value was obtained by log-rank test.
C6. Finally, is there a mechanistic explanation for the SDMA mark being a signal for protein degradation? Is there an E3 ligase that can read the SDMA marks on vimentin. This is a 8 critical missing link in this study.
R6: The reviewer's suggestion is well taken and we have included the potential explanation in the Discussion section. The related descriptions were included in the Discussion section (page 19) and marked in blue.
Page 19, line 9 to line 20 How can methylation modification be a signal for protein degradation? Since PTMs can lead to protein conformational change, the interaction accessibility of methylated proteins to E3 ligases or deubiquitinases would be altered (Yang et al, 2009). Two E3 ligases, TRIM16 and RNF208, were previously reported as negative regulators of vimentin (Marshall et al, 2010;Pang et al, 2019;Tian et al, 2020), but it is unclear whether their interaction with vimentin would be interfered by sDMA modification. Moreover, their targeting sites for ubiquitin conjugation on vimentin is outside of the coiled-coil rod domain, which is the major dimethylated region and responsible for arginine methylation-dependent ubiquitination in this study. The detailed regulation mechanisms could be elucidated by exploring the ubiquitome in the context of wild-type or 4RK vimentin. 9

Referee #2:
The authors identify the critical role MTAP/PRMT5 axis in post-transcriptional regulation of vimentin, which is a novel target for this post-translational modification/dimethylation. MTAP is responsible for catalyzing phosphorylation of MTA, so MTAP deficiency results in MTA accumulation, which reduces the level of PRMT5 (an arginine methyltransferase), which is required for demethylation of vimentin. This study uses methylproteomic screening to identify vimentin as novel dimethyl protein and cell and animal based assays with genetic knockout and post-transcriptional modification to test prove this mechanism of vimentin modification and its relevance to lung cancer progression. Overall, this study is very well written and the data very clearly illustrates the importance of their findings.
C1. In the abstract the authors say sDMA modification trivially affects the filamentous structure of vimentin and I would say this has not been fully proven, since only molecular methods were used to study vimentin structure. This is a small point that can be addressed through high resolution confocal microscopy experiments or other high-resolution imaging or structural analysis.

R1:
We would like to thank the reviewer for the constructive comments. In accordance with the suggestions, we have repeated the immunofluorescence experiments in order to confirm our findings and have revised Figures 4A and EV4A accordingly. The related methodology in the Materials and Methods section (page 27) has also been revised.
Page 29, line 6 to line 8 Fluorescent images were taken by a confocal microscope (Carl Zeiss LSM880, Oberkochen, Germany) with Airyscan detector and were processed by ZEISS ZEN2 image software.
Revised Figure 4A 10 A. Immunofluorescence staining of V5-vimentin wild-type, 4RK and 4RF mutants expressed in CL1-0 cells. F-actin was stained with phalloidin, and nucleus was stained with DAPI. Scale bars, 10 μm. Data shown are representative of three independent experiments.
Revised Figure EV4A A. Immunofluorescence staining of vimentin in indicated cell lines.  Fig. S2a?
R1: We thank the reviewer for the helpful suggestion. We have revised the Figure EV2A and related figure legends and marked in blue (page 49).
Revised Figure EV2A A. MTAP-mediated metabolic alterations in polyamine, methionine and adenine salvage pathways. The numbers are the average fold change intensities and associated errors for metabolites of CL1-5 MTAP/CL1-5 Mock (Student t test, n=3, biological replicates, *p < 0.05).
C2. Bottom of page 11: A more accurate statement would be that the residue identity at these methylation sites governs the migration promotion of vimentin since sDMA is not being directly measured but correlated. Thank you for submitting your revised manuscript. It has now been seen by one of the original referees.
As you can see, the referee finds that the study is significantly improved during revision and recommends publication. However, the referee has remaining concerns that need to be addressed prior to publication. Please repeat the experiment in Figure 2C and replace it with a higher quality blot as requested by the referee.
Moreover, I need you to address the editorial points below.
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Thank you again for giving us to consider your manuscript for EMBO Reports, I look forward to your minor revision. 1) The experiment depicted in Figure 2C was not repeated and replaced. As I said before, this is a key figure, and the quality of the data is extremely poor. There is a dark smudge over the part of the blot that they highlight as a SDMA signal on vimentin. Again, this experiment needs to be repeated and the quality of the blot improved.
2) Also, point #3 was not fully addressed. I asked the authors to remove all indication that F is a mimic for Rme2s. They have still included this misleading term on page11, line17. This must be removed.
The SYM10 antibody validation using vimentin peptides was nicely performed.

1
Comments from the Referee #1: Figure 2C was not repeated and replaced. As I said before, this is a key figure, and the quality of the data is extremely poor. There is a dark smudge over the part of the blot that they highlight as a SDMA signal on vimentin. Again, this experiment needs to be repeated and the quality of the blot improved.

C1. The experiment depicted in
R1: We would like to thank the reviewer for the suggestion. In accordance with the suggestion, we have repeated the in vitro methyltransferase assays and replaced Figure 2C with a higher quality blot as shown below.
Revised Figure 2C C. In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti-sDMA antibody. Arrow marks dimethyl-vimentin.
C2. Also, point #3 was not fully addressed. I asked the authors to remove all indication that F is a mimic for Rme2s. They have still included this misleading term on page11, line17. This must be removed.
R2: We thank the reviewer for the comment. We have accordingly revised the description for the 4RF mutant in the Results section (page 11) and marked in blue.
Page 11, line 15 to line 18 To investigate if the functionality of vimentin is mediated by its sDMA level, endogenous vimentin in H1650 and CL1-5 cells was knocked out by CRISPR/Cas9 approaches and the effect of wild-type, 4RK and 4RF (R196/207/345/364F, bulky hydrophobic form) mutant vimentin on cell motility was evaluated.
23rd May 2022 2nd Authors' Response to Reviewers 8th Jun 2022 2nd Revision -Editorial Decision Dear Sung-Liang, Thank you for submitting your revised manuscript. I have now looked at everything and all is fine. Therefore, I am very pleased to accept your manuscript for publication in EMBO Reports.
Congratulations on a nice work! Of note, I would like to remind you once again to make the dataset PXD031192 publicly available as soon as possible, which is prerequisite for publication. Thank you.
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